Project 1 will probe B-cell repertoires in the setting of influenza vaccination and infection to define pathways of dominant (monosubtypic) and subdominant (heterosubtypic) neutralizing antibody responses. The goal will be to design immunogens that have high affinity for reverted unmutated ancestors (RUAs) and intermediate antibodies of clonal lineages of broadly neutralizing heterosubtypic antibody producing B cells, in order to design immunogens that stimulate heterosubtypic dominant antibody responses. We hypothesize that comparison of plasma cell anti-HA antibody repertoires after TIV with repertoires from influenza-infected subjects, as well as analysis of inferred RUAs, will define the maturation pathways and novel influenza HA antigens that will that favor heterosubtypic antibody responses (or breadth within a subtype) and result in novel vaccine design. The hypotheses of the specific aims of this project are 1) Hypothesis: The B-cell repertoires elicited by non-replicating influenza vaccines and by infection differ in degree of polyclonal activation and breadth of neutralization. Moreover. the repertoires elicited by adjuvanted and non-adjuvanted vaccines differ because adjuvant broadens the range of recognized epitopes; 2) Hypothesis: Broadly reactive Abs, including those recognizing the heterosubtypic stem epitope, are less frequent after true primary than after secondary influenza immunization due to broadening of the response by multiple HA stimulations; and 3) Hypothesis: Efficient induction of Abs against a desired epitope requires: (a)proliferation of a favorable germline Ab and (b) an affinity maturation pathway to a desired final specificity. Thus, Project 1 will generate the Abs, analyze their clonal lineages, and express their inferred RUAs and inferred intermediate antibodies, not only for testing these hypotheses in Project 1 but as well for the work in Projects 2 and 3. Analysis of broadly neutralizing antibodies and definition of their maturation pathways will lead to design of immunogens that promote affinity maturation of otherwise subdominant, broadly neutralizing, heterosubtypic influenza HA antibody responses and lead to the development of an influenza vaccine that induces more subtypic or heterosubtypic breadth than currently available vaccines.